We stained 50mcl of whole human blood with anti-CD3 (SK7 clone, APC, BD biosciences). After 30 min of staining we added 1ml of x1 RBC buffer (42240, Biolegend) and incubated 15 min. The procedure of lysis of red blood cells we performed according manufacturer's instructions. After that cells were centrifuged (600g, 10min) and washed with 1ml PBS using the same speed (600g, 10min).
We analysed quantity of CD3+ cells on Accuri C6 plus. We detected 6,000 cells in 50mcl or 6,000x20= 120,000/ml.
According to the literature (ex: doi.org/10.1371/journal.pone.0097391) in 1ml of human blood there are 10 time more CD3+ cells: approx. 10^6cells/ml.
Could anybody faced such problem with Accuri C6?
Worth to note that device is installed properly and passed all CS&T and trucount tests.
Thanks a lot in advance!
The lymphocyte reference ranges percentages [absolute counts - Abs, cells/μl] unadjusted for gender differences for adolescents are: CD3: 49-83 [939-2959]; CD4: 27-53 [467-1563]; CD8: 16-40 [259-1262]; CD19+ B cells: 8-31 [169-1297] and CD16+CD56+ NK cells: 3-30 [59-1178] and for adults are: CD3: 65-88 [983-3572]; CD4: 26-62 [491-2000]; CD8: 14-44 [314-2,087]; CD19+ B cells: 2-27 [64-800] and CD16+CD56+ NK cells: 2-27 [27-693]. The ranges for CD4:CD8 ratio for adolescents and adults are 0.7-2.6 and 0.6-4.4, respectively. Gender based analysis of relative percentages of lymphocyte subsets showed no significant differences between adult and adolescent males and females. The mean CD4:CD8 ratio was significantly higher in adult females than males (P=0.04) and in adolescents this difference was not significant between genders. The mean CD3 and CD4 T cell percentages were higher and CD19 cell percentages were lower in adults compared to adolescents (P
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